Ran is a small, nuclear Ras-like GTPase that is required for nuclear transport, RNA transcription and processing, DNA replication, and cell cycle control. We have taken two approaches to the Ran GTPase pathway in Xenopus egg extracts: Purifying proteins that interact with Ran in a simple affinity assay, and cloning Xenopus homologues of known Ran-interacting proteins (Rips) in order to analyze their function in vitro. Using the first approach, we found that Ran associates specifically with at least ten extract proteins. We determined the identities of six Rips, and found that they include nuclear pore proteins and transport factors. We have examined the specificity of these interactions with respect to the guanine nucleotide bound to Ran and analyzed the interactions between endogenous Ran and Rips. We used the second approach to examine the role of RanBP1, a ubiquitous, highly conserved Ran binding protein, in nuclear assembly, transport and DNA replication. To do this, we removed RanBP1 from egg extracts by immunodepletion, and used the depleted extracts in nuclear assembly assays. We observed that RCC1, Ran's guanine nucleotide exchange factor (GEF), was unexpectedly co-depleted in these experiments. Our results suggest that neither RanBP1 nor RCC1 is essential for interphase nuclear functions in the absence of the other protein, but that the balance of RCC1 and RanBP1 is critical for proper nuclear assembly and function in vitro. Our work in Xenopus extracts is complemented by an analysis of S phase cell cycle controls in mammalian cells and by a search for the molecular target(s) of these controls. In particular, we have found mRNAs whose stability is controlled by the replication state of the nuclear DNA and we are in the process of characterizing these messages.